The main goals of this project are to determine how a novel pathway of ceramide metabolism to acylceramide (AC), recently elucidated by the PI, is regulated, and how this metabolic pathway leads to physical and functional sequestration of ceramide as AC in lipid droplets (LD) by the enzyme diacylglycerol-acyl-transferase (DGAT). An added goal is to uncover the implications of this ceramide metabolic pathway on regulation of cell biology and specifically cancer cell death. Work during this last cycle of the proposal uncovered a very novel and exciting pathway whereby we showed that ceramide synthases (CerSs) interact in a protein complex with acyl-CoA synthases (ACSLs) and DGATs to generate AC, which appears to reside in LD, thus sequestering ceramides (physically, and functionally) from being bioactive in cell stress pathways. This discovery in turn opened up a series of important questions about the role of this pathway in regulating sources and subcellular localization of bioactive ceramides that are acylated to AC, their role in regulating cell biology, and how to target this pathway for novel therapeutic approaches to cancer cell death. Taken together, we propose the following hypothesis: AC generation by the novel CerS/ACSL/DGAT pathway is dependent on specific subcellular pools of ceramides basally and in cell stress responses, and this metabolic pathway is a target for therapeutic development. As a corollary, DGATs, which function to acylate ceramide to AC, emerge as a key nodal point in regulating ceramide levels by forming AC. We wish to understand this metabolic pathway and define its significance. In order to address this hypothesis, we propose the following specific aims: Specific Aim 1. To determine the source and subcellular localization of ceramide incorporation into AC. The data from this aim should inform us on which specific sources of ceramide are substrates for AC formation and sequestration into LD for future therapeutic targeting. Specific Aim 2. To determine the biologic/cellular functions of the specific ceramides that are metabolized to AC in a DGAT dependent manner. These studies should define a role for DGATs as a major metabolic pathway in regulating bioactive pools of ceramide and cell stress responses. Specific Aim 3. To purify and determine the crystal structure of DGAT2 and determine its substrate specificity for ceramide vs diacylglycerol (DAG). These studies will advance a very novel concept relating to the metabolic regulation of AC and glycerolipids (triglycerides) by DGAT enzymes and their roles in cellular stress responses, a completely novel role for these important lipids in cell biology.